In the absence of an effective vaccine, alternative approaches to prevent sexual transmission of HIV-1 must be pursued. One such approach is the topical application of viral inhibitors prior to intercourse as a vaginal microbicide. We have recently demonstrated a proof-of-concept for this strategy using the rhesus macague vaginal transmission model and three inhibitory compounds: C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion, CMPD167, a small-molecule CCR5 ligand, and BMS-378806, a small molecule that binds gp120 and prevents its attachment to CD4. In vitro, all three compounds inhibit infection of T-cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, each compound can partially protect macaques from CCR5-using SHIV-162P3 infection, and C52L increases the protective capacities of CMPD167 and BMS-378806. The overall goal of this research plan is to identify and develop an optimized version of the recombinant C52L peptide fusion inhibitor as an inexpensive component of a topical HIV-1 microbicide formulation suitable for human testing. Our central hypothesis is that specific inhibitors of the virus-cell attachment and entry process, when used in combination, can help combat HIV-1 sequence diversity and minimize the transmission of variants resistant to any single inhibitor. Specific aims of this research are: (1) To identify and incorporate specific residue substitutions that optimize both potency and solubility of the C52L peptide inhibitor. Wewill use leucine- and glutamine-scanning mutagenesis to probe the contributions of specific side chains to fusion inhibition and aqueous solubility. We will also screen combinations of mutations in search of additive or synergistic effects. Our emphasis will be to piece together a new version of C52L with the optimal characteristics for topical formulations. (2) To evaluate the specificity, potency, and toxicity of improved C52L variants and their synergistic antiviral effects with CMPD167, BMS-378806, and the small- molecule CXCR4 inhibitor AMD3465 in vitro. We will conduct in vitro studies to determine the activity of select C52L variants against a diverse set of primary HIV-1 isolates, and their toxic or inflammatory effects using the rabbit vaginal irritation model and in human cells. We will also study antiviral synergy in vitro in order to make rational predictions for lead inhibitor combinations and formulations for in vivo efficacy testing in the macaque model. (3) To evaluate the protection of macaques from vaginal SHIV challenge by an optimized C52L product alone and in combination with CMPD167 and BMS-378806. We will use the rhesus macaque "high dose" vaginal transmission model with both CCR5 and CXCR4 SHIVs to assess the in vivo potency of the optimized C52L product alone and in synergistic combination with CMPD167 and BMS-378806.